A polymorphism is a variation in genetic sequence in which an individual's genetic sequence at a given location in the genome varies from the sequence commonly found in a population. Polymorphisms can be induced by mutagens, or they may be naturally occurring in a population. Presence of a polymorphism in an individual's DNA can often be used as a disease marker, since particular polymorphisms tend to be linked to certain diseases. Polymorphisms include single or multiple base substitutions, insertions or deletions. In human molecular and medical genetics, the vast majority of mutations and sequence polymorphisms in DNA result from single base substitution and small additions and deletions. The efficient, accurate and rapid detection of these variations is very important in the prediction and diagnosis of disease, forensic medicine, and public health.
Most methods for detection of genetic alternations consisting of one or a few bases involve hybridization between a standard nucleic acid (DNA or RNA) and a test DNA: the mutation may be revealed as a mispaired or unpaired base in a heteroduplex. Detection of these mispaired or unpaired bases has been accomplished by a variety of methods (Taylor, G. R., Laboratory Methods for the Detection of Mutations and Polymorphisms in DNA, CRC Press, (1997)), including denaturing gradient gel electrophoresis, enzyme or chemical mismatch cleavage, and direct sequencing of polymerase chain reaction products. These methods typically involve time-consuming or technically complicated methods that are not suitable for handling a large number of samples at one time, such as gel-electrophoresis and/or staining procedure. Some of the methods require that the exact location of the mutation be known, and the results can be difficult to interpret when the target DNA is heterozygous for the mutation in question. Thus, such techniques are not practical for use in screening of large numbers of samples for polymorphisms.
Other methods of detecting genetic alterations include chip- or sensor-based detection techniques, including methods using DNA microarrays or silicon-based DNA chips, and electrochemical, thermometric, microgivmetric, magnetic or optical methods, including fiberoptic methods. These techniques generally involve immobilization of a DNA probe strand to a support and subsequent hybridization of test DNA to the immobilized probe. Mutations that result in mispaired or unpaired bases in the hybridized heteroduplex molecule are detected by monitoring the hybridization affinity of the test DNA to the immobilized probe DNA, as these alternations affect the hybridization affinity (Knoll et al., Colloids and Surface A: Physicochemical and Engineering Aspects (2000) 169:137; Healy et al. Anal Biochem (1997) 251(2): 270-279) and thus produce different signal outputs, depending on the method use. The discrimination of the hybridization profiles of a mutant strand from wild-type DNA typically relies on the use of high stringency hybridization conditions, for example optimized hybridization temperature based on the melting properties (Tm) of the strands (Wittung-Stafshede P. et al., Colloid Surface A, 174: 269-273 (2000); Furtado, L. M. et al., Analyst, 123: 1937-1945 (1998)), high stringency buffer (Chong K. T. et al., Langmuir, 18:9932 (2003)), or both (Nilsson, P., et al, Laboratory Methods for the Detection of Mutations and Polymorphisms in DNA, Graham R. Taylor CRC Press, Boca Raton N.Y. 1997). These methods, however, are unsuitable for scanning wide regions of DNA (a capability essential for the detection of genomic polymorphisms) as single base substitutions or deletions result in very small differences in the hybridization affinity. As well, the detection sensitivity of these methods is in the level of the micromolar or sub-micromolar range, which is far below the limit required for disease diagnosis.
MutS, a DNA mismatch binding protein, has been used to detect mismatches in DNA samples. Wagner (U.S. Pat. Nos. 6,027,877, 6,114,115, and 6,329,147) describes methods in which labelled DNA is contacted with MutS protein for detection of any mismatches in the DNA sample. The MutS is immobilized on a chromatography column. Since these methods use chromatography methods, they are not well adapted to parallel analysis of multiple samples.
Thus, there is a need for a method of detecting mutations and polymorphisms in a DNA sample that is accurate, fast and easy to use, is suited for high-throughput analysis, and which enables identification of the location and nature of the mutation within a target DNA.